The present invention pertains to a composition comprising a combination formed by mixing two components: (1) a growth factor related to epithelial cell functions; and (2) an extracellular matrix-degrading protease for the purpose of enhancing healing of an injury in an animal, or human. The two components can be pre-combined before giving to the subject to be treated or given to the subject in a sequential manner. More specifically, this invention pertains to the combination of keratinocyte growth factors (“KGF”) with plasmin, plasminogen, plasminogen activator, or its functional biological equivalent, which combination is used for the treatment of injuries involving cells of epithelial origin and other cell types that KGF may affect. In addition, the present invention pertains to a kit that holds a container with a growth factor and a container with an extracellular matrix-degrading protease enzyme. The contents of each container can either be in a carrier solution (e.g. water, buffer, saline, thickener, emulsion, or ointment), or lyophilized with the carrier solution in a third container for the user to reconstitute the components. A further goal of the present invention is to present a method for using the components of the kit for the purpose of enhancing wound healing. For example, the components of the kit are mixed and placed on an injury, or the individual components of the kit can be placed on the injury in a tandem or sequential order. Alternately, the components or biological equivalents of the kit can be purchased or fabricated and used with the described methods for the purpose of enhanced wound healing.
Epithelial cells account for one-third of all cells in the human body. Those in skin are referred to as keratinocytes. Epithelial cells are also found as the surface lining in the mouth, nasal tract, gastrointestinal tract, vaginal tract, and several other organs or tissues such as lung and cornea. Injuries or wounds involving epithelial cells occur in many different forms, including cuts, burns, and ulcers. They may be acute or chronic. Examples include pressure ulcers, venous stasis ulcers, diabetic foot ulcers, duodenal ulcers, ulcerative colitis, aphthous ulcers, cornea ulcers. The non-healing and slow-healing wounds are referred as chronic wounds. Chronic wounds in the form of oral or intestinal mucositis occur frequently in cancer patients receiving chemotherapy. They are a major health concern and are increasingly so because of the aging population.
Endothelial cells are another large population of cells in the human body. They make up the surface lining of all blood vessels. The vasculature of a 70 kg adult is lined by ˜1,000 m2 of endothelial cells. In the aging human population, vascular damage related to heart disease, stroke, and lower extremity ischemia is one of the most significant medical concerns. Heart disease involving large blood vessels can be treated with certain surgical procedures such as angioplasty or by-pass surgery. However, no effective treatment is available for disease conditions involving small blood vessels. Recently, a promising new treatment for these vascular disease conditions, i.e., therapeutic angiogenesis, is being developed, which involves delivery of an angiogenic growth factor that promotes new blood vessel formation. A therapeutic mixture comprised of L-arginine and angiogenic growth factors is disclosed in U.S. Pat. No. 6,239,172 for the treatment of diseases related to endothelial dysfunction, the entire content of which is hereby incorporated by reference.
Extracellular matrix is a vital fibrous matrix providing support and anchorage for cells in establishing the tissue structure and integrity and maintaining their normal functions. Its components include collagen, fibronectin, laminin, heparin sulfate proteoglycan, hyaluronic acid, and elastin. In the case of wounds, blood clots formed by fibrin fibers in or on the damaged tissues constitute another matrix component. For cells to migrate through this matrix during wound healing or normal tissue growth, the matrix degradation surrounding the migrating cells is required. Although not wanting to be bound by theory, many enzymes are known to participate in this degradation process, including collagenases, metalloproteases, and plasmin. An intricate system exists to regulate the activities of these enzymes. Many of them are produced by cells as an inactive proenzyme requiring activation, e.g., by another enzyme, before they can be functional. Some of these enzymes have receptors on cell surfaces, thus limiting their activities near the cells.
KGF is a member of the fibroblast growth factor (“FGF”) family and is also known as fibroblast growth factor-7 (“FGF-7”) (Werner, Cytokine & Growth Factor Reviews 9:153–165, 1998). Members of this family are characterized by their ability to bind heparin, which plays a critical role in mediating the interaction between growth factors and their receptors (Schlessiger et al., Cell 83:357–360, 1995). The prototypes of the FGF family, aFGF (FGF-1) and bFGF (FGF-2), have been widely studied. KGF was first isolated in 1989. (Rubin et al., Proc. Natl. Acad. Sci. USA 86:802–806 (1989)). Subsequently, the KGF gene was cloned and KGF was expressed in bacteria. Finch et al., Science 245, 752–755 (1989); Ron et al., J. Biol. Chem. 268:2984–2988 (1993); Rubin et al., U.S. Pat. No. 5,741,642. It is unique in that it is produced by cells of mesenchymal origin, but primarily acts on cells of epithelial origin. KGF stimulates the proliferation and differentiation of epithelial cells (Aaronson et al., Annals of the New York Academy of Sciences 638, 62–77, 1991).
Mature KGF is a 163-amino acid polypeptide. Structural and mutagenesis studies have shown that the heparin and receptor-binding domains are located near the center portion of the molecule. A KGF fragment consisting of residues 23–144 (Δ23 KGF-R144Q) retains the original biological activity along with increased thermal stability. (Osslund et al., Protein Science 7:1681–1690 (1998)). U.S. Pat. No. 5,677,278 (“the '278 patent”) discloses a truncated keratinocyte growth factor fragment (Δ23 KGF or KGF lacking the first 23 amino acids from N-terminus) which exhibits at least a 2-fold increase in mitogenic activity as compared to a mature full-length keratinocyte growth factor. The '278 patent further relates to a conjugate of truncated KGF and a toxin molecule for use in treatment of hyperproliferative disease of the epidermis. Moreover, the '278 patent relates to a therapeutic composition containing truncated KGF and a pharmaceutically acceptable carrier and the use thereof for wound healing purposes. The entire content of the '278 patent is hereby incorporated by reference.
There are four types of FGF receptors (“FGFR”), FGFR1-FGFR4. Several isoforms of FGFRs have been identified. While aFGF binds to all four types of FGFRs, KGF is only known to bind to the IIIb isoform of FGFR2. KGF is highly expressed in skin wounds. Increased KGF expression has also been observed in the bladder and kidney following injury and in inflammatory bowel disease. Unlike other growth factors, KGF is persistently expressed at a high level during the course of healing (Werner et al., Proc. Natl. Acad. Sci. USA 89, 6896–6900, 1992). Animal studies have shown that applying KGF topically to a wound accelerates the healing. (Staiano-Coico et al., J. Exp. Med. 178:865–878 (1993); Pierce et al., J. Exp. Med. 179: 831–840 (1994); Wu et al., Arch. Surg. 131:660–666 (1996)). Systemic administration of KGF has been shown to reduce injury in experimentally induced colitis. KGF also protects epithelial cells in animals subjected to radiation and/or chemotherapy. Furthermore, glucocorticoid treatment, which is known to delay wound healing, suppresses KGF expression. The use of compositions and devices for the controlled release delivery of peptides and growth factors have been disclosed in U.S. Pat. No. 6,187,330 (“the '330 patent”). The '330 patent invention may be employed for local delivery of angiogenic basic fibroblast growth factor or vascular endothelial growth factor, and the entire content is hereby incorporated by reference.
In U.S. Pat. No. 5,965,530 (“the '530 patent”) KGF was shown to act on other types of cells, and the entire content is hereby incorporated by reference. Based on extensive in vivo studies in animals, it has now been discovered that KGF stimulates proliferation, growth and differentiation of various types of cells, besides keratinocytes. This better understanding of the biological effects of KGF in vivo enables a wider use of this polypeptide as a therapeutic agent, suitably formulated in a pharmaceutical composition, for the specific treatment of disease states and medical conditions afflicting or affecting tissues and organs such as the dermal adnexae, the liver, the lung, and the gastrointestinal tract. Besides cells of epithelial origin, KGF has recently been shown to act on microvascular endothelial cells, but not those from large vessels such as the aorta. It stimulates chemotaxis and proliferation of microvascular endothelial cells and induces angiogenesis in the rat cornea. Gillis et al., Journal of Cell Science 112:2049–2057 (1999).
A growth factor similar to KGF has recently been identified, named FGF-10 or KGF-2 (Yamasaki et al., Journal of Biological Chemistry 271:15918–19521 (1996); Beer et al., Oncogene. 15:2211–2218 (1997); Igarashi et al., Journal of Biological Chemistry. 273:13230–13235 (1998); Jimenez and Rampy, Journal of Surgical Research. 81:238–242 (1999)). It is in the same FGF family and shares a 54% amino acid identity with KGF. It also acts primarily on epithelial cells. However, unlike KGF, FGF-10 also binds FGFR1 and binds more strongly to heparin. FGF-10 does not appear to be as highly expressed as KGF in skin wounds, although topical application of FGF-10 has been found to enhance the healing process.
Besides KGF and FGF-10, there are other growth factors that can stimulate epithelial cells. These growth factors include epidermal growth factor (“EGF”), hepatocyte growth factor (“HGF”), transforming growth factor-α (“TGF-α”), insulin-like growth factor I (“IGF-I”), and acidic fibroblast growth factor (“aFGF”). However, these growth factors also act on other types of cells such as fibroblasts, hepatocytes, and muscle cells.
The migration of epithelial cells, like that of many other cells, is thought to be closely associated with the expression of urokinase plasminogen activator (“uPA”) and its receptor (“uPAR”) (Morioka et al., J. Invest. Dermatol. 88:418–423, 1987; Romer et al., J. Invest. Dermatol. 102:519–522, 1994). The role of uPAR is to focus the uPA-mediated fibrinolysis or proteolysis on the cell surface. The uPAR binds the single-chain inactive uPA or scuPA, which is then converted to the active two-chain uPA. KGF has been shown to increase the expression of uPA in epithelial cells (Tsuboi et al., J. Invest. Dermatol. 101:49–53, 1993; Zheng et al., European Journal of Cell Biology 69:128–134, 1996). uPA converts plasminogen to plasmin. Plasminogen is a ˜90 kDa glycoprotein and consists of five kringle domains and a protease domain. Activation occurs by cleavage of plasminogen at a single peptide bond (Arg 561-Val 562). The two chains remain linked together by a disulfide bond following activation. Plasminogen also binds to cell surfaces via plasminogen binding sites, which is then readily activated by uPAR-bound uPA. Tissue plasminogen activator (tPA) also activates plasminogen, but does not have a cell surface receptor and requires binding to fibrin for optimal enzyme activity. Plasminogens lacking the first four or all five kringle domains are referred to as mini-plasminogen or micro-plasminogen. They can be similarly activated as plasminogen. Komorowicz et al., Biochemistry 37:9112–9118 (1998).
Plasmin/plasminogen is the key enzyme in fibrinolysis (Collen, Thrombosis and Haemostasis 82:259–270, 1999). It is responsible for degrading fibrin clots and other extracellular matrix components, but it is also involved in activation of metalloproteases and growth factors such as TGF-β. Besides uPA and tPA, plasminogen can also be activated by other enzymes, including streptokinase, staphylokinase, and common vampire bat plasminogen activator. Recent studies have shown that plasmin is critical for wound healing. Mice with a plasminogen gene knock-out exhibited delayed skin wound healing as the result of excess fibrin deposition, which in turn prevents keratinocyte migration (Romer et al., Nature Medicine 2: 287–292, 1996). If the fibrinogen gene is also deleted in these mice, wound healing ability is rescued (Bugge et al., Cell 87:709–19, 1996). The importance of plasmin has also been observed in the healing of artery and corneal injuries (Carmeliet et al., Circulation 96:3180–91 (1997); Kao et al., Investigative Ophthalmology & Visual Science 39:502–508 (1998)). In chronic venous leg ulcers, a defect in plasminogen has been observed (Hoffman et al., J. Invest. Dermatol. 111: 1140–4, 1998). Furthermore, deletion of uPA and/or tPA gene also causes delayed wound healing as a result of excess fibrin deposition. (Bugge et al., Proc. Natl. Acad. Sci. USA 93:5899–5904 (1996); Heymans et al., Nature Medicine. 5:1135–1142 (1999)). The plasmin has been used as a topical agent for treatment of wounds and has also been evaluated as an injectable for treatment of thrombosis.
The initial step of angiogenesis includes the migration of endothelial cells, and also involves proteolytic degradation of the extracellular matrix. The expression of uPA and uPAR is upregulated in the migrating endothelial cells (Carmeliet and Collen, Trends in Cardiovascular Medicine 7:271–281, 1997). Although not wanting to be bound by theory, the plasminogen activators and plasminogen trigger a protease cascade that may play an important role in angiogenesis. Studies using gene knock-out mice have shown that uPA-deficient mice exhibited an impaired revascularization, even after the treatment with vascular endothelial growth factor (VEGF), a known angiogenic growth factor (Heymans et al., Nature Medicine. 5:1135–1142, 1999).
Besides plasmin/plasminogen, there are other enzymes that may be involved in fibrin degradation. One such class of enzyme is metalloprotease (MMP), which has over 20 members. MMP-12 and -14 are known to degrade fibrin. The healing of wounds on plasminogen-deficient mice was severely delayed, but the wounds eventually healed. In contrast, if a broad-spectrum MMP inhibitor (e.g. galardin) was applied to these mice, the healing of wounds was completely stopped (Lund et al., EMBO J., 18, 4645–4656, 1999). The degradation of fibrin by MMP-14 also is involved in endothelial cell migration (Hiraoka et al., Cell, 95, 365–377, 1998).
The use of both thrombin inhibitors and recombinant plasminogen activators have been used in products to control the wound healing processes. For example, U.S. Pat. No. 6,174,855 disclose the use of a thrombin inhibitor in the manufacture of a product for use in the control of wound healing processes within the body, in particular, the inhibition or prevention of fibrin-related adhesion and/or scar tissue formation, as well as products for use in the control of wound healing processes within the body comprising polysaccharides (e.g., chitosans) and low molecular weight peptide-based thrombin inhibitors, and the entire content of which is hereby incorporated by reference. Additionally, U.S. Pat. No. 6,033,664 (“the '664 patent”) discloses the use of non-bacterial plasminogen activators in the manufacture of a topical medical preparation for the treatment of slow- or non-healing wounds. In addition, the '664 patent relates to a composition comprising a physiologically acceptable carrier and an effective amount of a non-bacterial plasminogen activator, with the exclusion of tPA, and the entire content of which is hereby incorporated by reference.
Despite all advances, there is still a great need for agents that can effectively treat injuries involving cells of epithelial origin. As previously stated, prior art teaches that injuries can be treated with various mixtures of protein-derived growth factors or protein growth factors. Additionally, prior art teaches that a slow healing injury can be treated with proteolytic enzymes. The combined use of protein growth factors and proteolytic enzymes to aid in wound healing are counter-intuitive, even though the functions of the protein growth factors and the enzyme may seem to be complimentary. The most obvious reason for such reasoning is that proteolytic enzymes function to hydrolyze (digest) proteins. Thus, the combination of protein growth factors (e.g. KGF), and enzymes that digest proteins (e.g. plasminogen) would initially seem counter-productive as a treatment for wound healing to one with ordinary skill in the art. However, insight is provided in the present invention that suggests a synergistic effect by the combination of growth factors and proteolytic enzymes. It is particularly desired that such combinatorial agents are produced and used based on their intricate interaction under in vivo conditions.